Yersinia pestis, the etiological agent of the acute diseases bubonic and pneumonic plague, has been one of the most devastating epidemic-causing bacteria experienced by mankind. Although plague is not a public heath problem in most parts of the world, its potential for contagion, the lack of an effective vaccine, and the recent emergence of multiple antibiotic resistant strains place this organism squarely at the top of the United States' select agent list as a potential candidate for bioterrorism use. The long-term goal of this application is to elucidate the molecular mechanisms that underlie the nature of the acute bacterial infectious process in Y. pestis. The more immediate objective of this work is to identify novel virulence genes that will serve as targets in the development of robust diagnostics assays (especially for genetically-engineered organisms) and of non-antibiotic therapeutics. Specific aims of this application are as follows. Specific Aim 1 is to determine the role played by Y. pestis-specific genomic regions in its pathogenicity by generating systematic knockouts of these genomic regions. These unique regions will be identified by direct, whole-genome comparison between Y. pestis and its enterophathogen progenitor, Y. pseudotuberculosis, while the putative change in virulence will be determined using a murine model of infection. Specific Aim 2 is to evaluate the contribution to virulence of Y. pestis genes differentially expressed in relation to its near-neighbor, Y. pseudotuberculosis. This will be done by first identifying the differentially-expressed genes using an available whole-genome microarray of the two Yersinae spp. followed by generation of knockouts as described for Specific Aim 1. Specific Aim 3 is to characterize the genome-wide expression profile of attenuated mutants obtained in Specific Aims 1 and 2 in order to identify the relevant virulence pathways of this organism. This will be achieved using similar microarray analyses. The outlined application will serve as an important first step in elucidating the genomic basis for the dramatically different clinical manifestations of these two genetically related pathogens. This work will provide a unique model for defining the nature of this and other acute infectious diseases through the use of recent advances in comparative and functional genomics. Similar approaches can be used to study the virulence of other potential bacterial bioterrorism agents.